SARS-CoV-2 Breakthrough Infections after Third Doses Boost IgG Specific Salivary and Blood Antibodies.

SARS-CoV-2 breakthrough infections, associated with waning immunity, increase systemic antibody levels. In this study, we analyzed the impact of the infection timing on the magnitude of the systemic humoral response and whether breakthrough infections also boost antibody levels in the salivary compartment. We observed that the combination of infection plus vaccination, regardless of infection timing, produced a sharp increase in systemic antibodies, which were higher in subjects infected after third doses. Moreover, despite high systemic antibody levels, breakthrough infections after dose three occurred and boosted antibody levels in the salivary compartment. These results suggest that current vaccination strategies against COVID-19 should be improved. Results also showed that determination of salivary antibodies against SARS-CoV-2 could be a valuable tool in disease prevalence studies, for the follow-up of vaccinated individuals, and to assist vaccination strategies against COVID-19, especially in settings where blood sampling cannot be fulfilled.

[Alterations of immunological parameters in an autochthonous case of visceral leishmaniasis from the Northwest of Argentina]. / Afectación de parámetros inmunológicos en un caso de leishmaniasis visceral autóctono del Noroeste Argentino.

We report the alterations of immunological parameters of a patient with visceral leishmaniasis caused by Leishmania (Leishmania) infantum from the Northwest of Argentina during active disease and after achieving clinical recovery. We first demonstrated elevated amounts of IFN-y, IL-10, B-cell activating factor (BAFF) and IgG in plasma during active disease, which returned to control values after recovery. In relation to T cell profile, we measured CD27, CD28, CD45RO, CD57 and perforin. We found a highly differentiated phenotype, preferentially in active disease and among CD8+ T cells, consisting in increased numbers of late differentiated and terminal effector cells. Although this highly differentiated CD8+ T cell phenotype persisted after recovery, a clear increase of central memory cells was recorded for both T subsets at that point, suggesting signs of reversion toward a less differentiated profile. The composition of the B cell compartment was slightly modified during active disease. Herein we document the global impact of severe visceral leishmaniasis on immunological parameters, which tend to revert upon clinical recovery, suggesting signs of immune restoration accompanying clinical improvement. The evaluated parameters could eventually be used as biomarkers of clinical evolution of visceral leishmaniasis.

En el presente trabajo informamos la afectación de parámetros inmunológicos durante la etapa grave de la infección y luego de alcanzar la recuperación clínica en un paciente autóctono del Noroeste argentino con leishmaniasis visceral causada por Leishmania (Leishmania) infantum. Detectamos concentraciones plasmáticas elevadas de interferón-y, interleuquina 10, IgG y BAFF (B-cell activating factor) durante la enfermedad activa, que se normalizaron luego de la recuperación clínica. En relación al perfil de diferenciación y memoria de las células T, clasificamos las células según la expresión de CD27, CD28, CD45RO, CD57 y perforina. Encontramos un fenotipo altamente diferenciado analizando la población de linfocitos T CD8+, con porcentajes aumentados de células T de diferenciación tardía y efectoras terminales. Si bien el fenotipo T CD8+ persistió luego de la recuperación clínica, pudimos observar un claro aumento de células T de memoria central en ese punto de estudio, sugiriendo signos de una posible reversión hacia un perfil T menos avanzado. El compartimiento de células B CD19+ mostró cambios más leves en la composición de las subpoblaciones de memoria. Documentamos el compromiso global de parámetros inmunológicos en la etapa grave de la leishmaniasis visceral que tienden a revertir luego de la recuperación, sugiriendo posibles signos de reconstitución inmune acompañando a la mejoría clínica. Los parámetros evaluados podrían ser útiles como biomarcadores de la evolución clínica de la enfermedad.

Afectación de parámetros inmunológicos en un caso de leishmaniasis visceral autóctono del Noroeste Argentino / Alterations of immunological parameters in an autochthonous case of visceral leishmaniasis from the Northwest of Argentina

Resumen En el presente trabajo informamos la afectación de parámetros inmunológicos durante la etapa grave de la infección y luego de alcanzar la recuperación clínica en un paciente autóctono del Noroeste argentino con leishmaniasis visceral causada por Leishmania (Leishmania) infantum. Detectamos concentraciones plasmáticas elevadas de interferón-γ, interleuquina 10, IgG y BAFF (B-cell activating factor) durante la enfermedad activa, que se normalizaron luego de la recuperación clínica. En relación al perfil de diferenciación y memoria de las células T, clasificamos las células según la expresión de CD27, CD28, CD45RO, CD57 y perforina. Encontramos un fenotipo altamente diferenciado analizando la población de linfocitos T CD8+, con porcentajes aumentados de células T de diferenciación tardía y efectoras terminales. Si bien el fenotipo T CD8+ persistió luego de la recuperación clínica, pudimos observar un claro aumento de células T de memoria central en ese punto de estudio, sugiriendo signos de una posible reversión hacia un perfil T menos avanzado. El compartimiento de células B CD19+ mostró cambios más leves en la composición de las subpoblaciones de memoria. Documentamos el compromiso global de parámetros inmunológicos en la etapa grave de la leishmaniasis visceral que tienden a revertir luego de la recuperación, sugiriendo posibles signos de reconstitución inmune acompañando a la mejoría clínica. Los parámetros evaluados podrían ser útiles como biomarcadores de la evolución clínica de la enfermedad.

Abstract We report the alterations of immunological parameters of a patient with visceral leishmaniasis caused by Leishmania (Leishmania) infantum from the Northwest of Argentina during active disease and after achieving clinical recovery. We first demonstrated elevated amounts of IFN-γ, IL-10, B-cell activating factor (BAFF) and IgG in plasma during active disease, which returned to control values after recovery. In relation to T cell profile, we measured CD27, CD28, CD45RO, CD57 and perforin. We found a highly differentiated phenotype, preferentially in active disease and among CD8+ T cells, consisting in increased numbers of late differentiated and terminal effector cells. Although this highly differentiated CD8+ T cell phenotype persisted after recovery, a clear increase of central memory cells was recorded for both T subsets at that point, suggesting signs of reversion toward a less differentiated profile. The composition of the B cell compartment was slightly modified during active disease. Herein wedocument the global impact of severe visceral leishmaniasis on immunological parameters, which tend to revert upon clinical recovery, suggesting signs of immune restoration accompanying clinical improvement. The evaluated parameters could eventually be used as biomarkers of clinical evolution of visceral leishmaniasis.

B-cell profile, B-cell activating factor concentration and IgG levels in human cutaneous and mucosal leishmaniasis.

AIMS: The aim of this study was to evaluate characteristics of B cells in human tegumentary leismaniasis (TL) analysing cutaneous leishmaniasis (CL), most prevalent form and mucosal leishmaniasis (ML), aggressive form characterized by the destruction of the oral-nasal-pharyngeal cavities. METHODS AND RESULTS: By flow cytometry analysis, we found decreased percentages of non-class-switched memory B cells in TL with the degree of the loss related to clinical severity. Using commercial ELISA, we reported high levels of B-cell activating factor (BAFF) and IgG preferentially in aggressive CL and markedly in ML together with decreased BAFF receptors in the latter. We also found lower levels of BAFF after clinical recovery suggesting a relation between BAFF and disease activity. Mucosal leishmaniasis history of therapeutic failure presented high levels of BAFF accompanied by detectable concentrations of IFN-γ and IL-6 (assayed by commercial ELISA and cytometric bead arrays respectively), cytokines involved in exaggerated inflammatory responses and tissue damage in TL. CONCLUSION: We demonstrate B-cell disturbances in TL with the degree of the alterations related to clinical severity. We suggest a relation between excess of BAFF and disease activity and point towards a possible implication of BAFF in the inflammatory phenomenon of ML.

American tegumentary leishmaniasis: T-cell differentiation profile of cutaneous and mucosal forms-co-infection with Trypanosoma cruzi.

American tegumentary leishmaniasis displays two main clinical forms: cutaneous (CL) and mucosal (ML). ML is more resistant to treatment and displays a more severe and longer evolution. Since both forms are caused by the same Leishmania species, the immunological response of the host may be an important factor determining the evolution of the disease. Herein, we analyzed the differentiation and memory profile of peripheral CD4(+) and CD8(+) T lymphocytes of patients with CL and ML and their Leishmania-T. cruzi co-infected counterparts. We measured the expression of CD27, CD28, CD45RO, CD127, PD-1 and CD57, together with interferon-γ and perforin. A highly differentiated phenotype was reflected on both T subsets in ML and preferentially on CD8(+) T cells in CL. A positive trend toward a higher T differentiation profile was found in T. cruzi-infected CL and ML patients as compared with Leishmania single infections. Association between CD8(+) T-cell differentiation and illness duration was found within the first year of infection, with progressive increase of highly differentiated markers over time. Follow-up of patients with good response to therapy showed predominance of early differentiated CD8(+) T cells and decrease of highly differentiated cells, while patients with frequent relapses presented the opposite pattern. CD8(+) T cells showed the most striking changes in their phenotype during leishmaniasis. Patients with long-term infections showed the highest differentiated degree implying a relation between T differentiation and parasite persistence. Distinct patterns of CD8(+) T differentiation during follow-up of different clinical outcomes suggest the usefulness of this analysis in the characterization of Leishmania-infected patients.

Common Variable Immunodeficiency and Circulating TFH.

CD4+ T follicular helper cells (TFH) were assessed in adult patients with common variable immune deficiency (CVID) classified according to the presence of granulomatous disease (GD), autoimmunity (AI), or both GD and AI (Group I) or the absence of AI and GD (Group II). TFH lymphocytes were characterized by expression of CXCR5 and PD-1. TFH were higher (in both absolute number and percentage) in Group I than in Group II CVID patients and normal controls (N). Within CXCR5+CD4+ T cells, the percentage of PD-1 (+) was higher and that of CCR7 (+) was lower in Group I than in Group II and N. The percentages of Treg and TFH reg were similar in both CVID groups and in N. TFH responded to stimulation increasing the expression of the costimulatory molecules CD40L and ICOS as did N. After submitogenic PHA+IL-2 stimulation, intracellular expression of TFH cytokines (IL-10, IL-21) was higher than N in Group I, and IL-4 was higher than N in Group II. These results suggest that TFH are functional in CVID and highlight the association of increased circulating TFH with AI and GD manifestations.

CD4+ T Lymphocytes with follicular helper phenotype (T(FH)) in patients with SH2D1A deficiency (XLP).

Peripheral blood mononuclear cells with T(FH) phenotype from two asymptomatic XLP patients were studied. Normal/high numbers of CXCR5+, CD4+ T cells coexpressing PD-1 were demonstrated. Peripheral blood mononuclear cells (PBMC) from these patients responded to sub-optimal PHA/IL-2 stimulation upregulating ICOS and CD40L and increasing intracellular expression of IL-10, IL-21 and IL-4 by CD4+ T(FH) cells. However when compared to N, the time profile of activation and cytokine synthesis was different in XLP and N. While ICOS and CD40L expression in N decreased after 6-8 days, it continued to increase or was maintained in XLP cultures. Intracellular IL-10, IL-21 and IL-4 reached higher values in XLP than N after 8 days. Rather than the absence of T(FH) cells or their intrinsic inability to respond to stimuli, differences in the time profile of their response could contribute to impair their role as helpers of B lymphocytes.

Ausencia del receptor CD16b en neutrófilos / Neutrophils without CD16b receptors

Se presentan dos pacientes (mujeres de 41 y 15 años de edad) con ausencia del receptor para el fragmento Fc de IgG, CD16b en neutrófilos (fenotipo "null"). El caso 1 fue referida al laboratorio con diagnóstico de hemoglobinuria nocturna paroxística y el caso 2) con diagnóstico presuntivo de neutropenia inmune. En ambos casos se comprobó por citometría de flujo la ausencia de expresión de CD16b, sin deficiencias en la expresión de otras moléculas del sistema de alloantígenos propios de neutrófilos ni defectos en el anclaje a membrana por glicosil fosfatidil inositol (GPI). Las manifestaciones clínicas en ambas pacientes: anemia en el caso 1 y leucopenia en el caso 2 no pueden ser atribuidas exclusivamente a la carencia de CD16b, ya que otros receptores para Fc de IgG (CD32 y CD64) podrían suplir la función de CD16b. Sin embargo, es importante tener en cuenta esta rara deficiencia (b y neutropenia isoinmune natal transitoria en niños nacidos de mujeres con fenotipo "null".

Occurrence of the rare CD16b deficiency ("null" phenotype) in neutrophils from two female patients (41 and 15 years old) is reported. The first case was referred with a diagnosis of anemia related to paroxistic nocturnal hemoglobinuria and the second case, with presumptive diagnosis of immune neutropenia. In both cases, absence of CD16b expression was determined by flow cytometry without deficiencies of other neutrophil alloantigens or defects of membrane anchorage through glycosil phosphatydil inositol (GPI) linkage. Clinical manifestations in both patients could not be attributed exclusively to the absence of CD16b, as other receptors for the IgG Fc fragment (CD32 and CD64) could compensate this deficiency that occurs in < 1% of the caucasic population. Nevertheless, it is important to take this rare deficiency into account in order to prevent isoantibody formation after eventual blood transfusions, or transient neonatal immune neutropenia in children born to women with the "null" phenotype.

[Neutrophils without CD16b receptors]. / Ausencia del receptor CD16b en neutrófilos.

Occurrence of the rare CD16b deficiency ("null" phenotype) in neutrophils from two female patients (41 and 15 years old) is reported. The first case was referred with a diagnosis of anemia related to paroxistic nocturnal hemoglobinuria and the second case, with presumptive diagnosis of immune neutropenia. In both cases, absence of CD16b expression was determined by flow cytometry without deficiencies of other neutrophil alloantigens or defects of membrane anchorage through glycosil phosphatydil inositol (GPI) linkage. Clinical manifestations in both patients could not be attributed exclusively to the absence of CD16b, as other receptors for the IgG Fc fragment (CD32 and CD64) could compensate this deficiency that occurs in <1% of the caucasic population. Nevertheless, it is important to take this rare deficiency into account in order to prevent isoantibody formation after eventual blood transfusions, or transient neonatal immune neutropenia in children born to women with the "null" phenotype.

Increased lymphocyte viability after non-stimulated peripheral blood mononuclear cell (PBMC) culture in patients with X-linked lymphoproliferative disease (XLP).

Survival of lymphocytes after prolonged culture was studied in two asymptomatic XLP patients. Viability of XLP PBMC after 30 days of non-stimulated culture was higher than that of normal controls (N), mainly due to the persistence of CD8 memory lymphocytes. IFNgamma high CD8 T lymphocytes remained higher in XLP than in N after 30 days. The number of perforin+ CD8 lymphocytes was markedly reduced after 30 days in XLP and in N. Increased viability was not related to CD127, PD-1, CD27, or CD62L expression. Concerning B lymphocytes, memory CD27+ CD19+ cells prevailed over CD27- cells after 30 days in both XLP and N, with far more surviving cells in XLP. In N, few CD19+ B lymphocytes were viable after prolonged culture. In XLP, these cells were also IgD+, IgM+ and EBNA2+. These results demonstrate that IFNgamma-positive memory CD8 T cells persist in XLP after prolonged culture in association with a subset of viable memory CD27+ B cells expressing latent EBV antigens. The survival advantage of XLP cells might be related to increased frequency of extranodal lymphoma in XLP patients.

Factors involved in the generation of memory CD8+ T cells in patients with X-linked lymphoproliferative disease (XLP).

UNLABELLED: We have analysed the phenotype of T lymphocytes in two X-linked lymphoproliferative disease (XLP) patients with the same SH2D1A mutation differing in initial exposure to Epstein-Barr virus (EBV) and treatment. While memory T lymphocytes (with low CCR7 and CD62L expression) prevailed in both XLP patients, in patient 9, who developed acute infectious mononucleosis (AIM) and received B cell ablative treatment, the predominant phenotype was that of late effector CD8 T cells (CD27-, CD28-, CCR7-, CD62L-, CD45 RA+, perforin+), while in patient 4 (who did not suffer AIM) the prevalent phenotype of CD8 T lymphocytes was similar to that of normal controls (N) or to that of adult individuals who recovered from AIM: CD27+ , CD28+, CCR7-, CD62L-, CD45 RO+ and perforin-. CD57 expression (related to senescence) was also higher in CD8 T cells from patient 9 than in patient 4, AIM or N. Persistently high EBV viral load was observed in patient 9. The results obtained from this limited number of XLP patients suggest that events related to the initial EBV encounter (antigen load, treatment, cytokine environment) may have more weight than lack of SH2D1A in determining the long-term differentiation pattern of CD8 memory T cells.

Impact of human immune deficiency virus infection on hepatitis C virus infection and replication.

Human immune deficiency virus (HIV) and human hepatitis C virus (HCV) infection are frequent in patients who have been exposed to blood or blood-derived products. It has been suggested that HIV infection increases HCV replication altering the course of HCV-related disease. However, it is not known if HIV directly enhances HCV replication or if its effect is the consequence of HIV infection of other cell types that control HCV replication (lymphocytes, macrophages). While the main cell targets for HIV infection are mononuclear leukocytes bearing CD4 and the chemokine receptors CCR5 and CXCR4, HCV was originally thought to be strictly hepatotropic, but it is now known that HCV can also replicate in peripheral blood mononuclear cells (PBMC). Therefore, in co-infected individuals, these two different viruses could share cell targets and interact either directly or indirectly. Some membrane receptors can be used by both HCV and HIV for entry into target cells, but the intracellular mechanisms shared by these viruses are not known. Lack of experimental systems providing suitable methods for the study of HCV replication in the presence or absence of HIV co-infection has hampered advances in this research area, but recent investigations are currently going on in order to answer these questions. This is an important issue, as knowledge of HIV/HCV interactions is required for the design of effective antiviral therapies.

[Autoimmune-alloimmune neonatal neutropenia. Serum reactive IgG and neutrophil-specific phenotype detected by flow cytometry]. / Neutropenia inmune-aloinmune neonatal. IgG sérica reactiva y fenotipo específico de los neutrófilos evaluados por citometría de flujo.

Auto or alloantibodies reactive with neutrophils define immune neutropenia. Alloimmune neonatal neutropenia is caused by maternal sensitization to paternal neutrophil antigens, resulting in IgG antibodies that are transferred to the fetus through the placenta. We present the studies in 4 children from 3 families with neutropenia of unknown origin (two of them were brothers). They were evaluated by flow cytometry in parallel with leukoagglutination. Reference values were established for serum reactive IgG in healthy volunteers for three dilutions (1/2, 1/5 and 1/20), both for the autologous reaction (serum and cells of the same individual) and for the heterologous reaction (serum and cells of different individuals). Results were expressed by an index defined by the quotient of the mean fluorescence intensity of the patient's serum divided by that of the reference serum. Serum reactive/agglutinant factors and circulating immune complexes were evaluated in patients and parents serum. Neutrophil specific phenotypes were determined for HNA-1a, HNA-1b and HNA-2a. Reactive IgG/agglutinant factors were found in 4 children. Two maternal sera were reactive against paternal and/or children neutrophils. Circulating immune complexes were detected in 2/4 children sera and were negative in 3/3 maternal sera. Maternal/children incompatibility was detected in the four cases. The three mothers had the same phenotype: homozygous NA1/NA1, NB1+.

Neutropenia inmune - aloinmune neonatal IgG serica reactiva y fenotipo especifico de los neutrofilos evaluados por citometria de flujo / Autoimmune-alloimmune neonatal neutropenia. Serum reactive IgG and neutrophil-specific

La neutropenia inmune se diagnostica por la presencia de auto o aloanticuerpos reactivos con losneutrófilos. La neutropenia aloinmune neonatal es consecuencia de la sensibilización materna alos antígenos específicos de los neutrófilos paternos que afectan al neonato al atravesar la barrera placentaria. Se presentan 4 casos de niños, 2 de ellos hermanos consanguíneos con doble vínculo. Se estudiaron los sueros de los pacientes y sus padres. Por citometría de flujo se establecen los valores de referencia de la IgG sérica reactiva con los neutrófilos en voluntarios sanos, para 3 diluciones (1/2, 1/5 y 1/20) en reacción autóloga(suero y células de un mismo individuo) y heteróloga (suero y células de diferentes individuos). Los resultadosse expresan por un índice definido como el cociente entre la mediana de la intensidad de fluorescencia media del suero incógnita y la de un suero utilizado como referencia. Por leucoaglutinación se evaluó la dilucióndel suero 1/20. Se determinó el nivel de complejos inmunes circulantes. Se determinó el fenotipo, para los epitopes HNA-1a, HNA-1b y HNA-2a. En los 4 niños se encontró IgG reactiva y/o factores aglutinantes; 2/3 sueros maternos fueron reactivos con los neutrófilos del cónyuge y de los hijos. Los complejos inmunes circulantes fueron positivos en 2/4 sueros negativos en 3/3 sueros maternos. Se encontró incompatibilidad materno-infantil en los 4 casos. Las 3 madres tenían igual fenotipo: homocigotos NA1/NA1, NB1+. En síntesis, se presenta el hallazgo de 4 casos con neutropenia inmune: 3/4 auto-inmune, 1/3 se asocia a complejos inmunes circulantes y 1/4 con neutropenia neonatal aloinmune (AU)

Auto or alloantibodies reactive with neutrophils define immune neutropenia. Alloimmune neonatal neutropenia is caused by maternal sensitization to paternal neutrophil antigens, resulting in IgG antibodies that are transferred to the fetus through the placenta. We present the studies in 4 children from 3 families with neutropenia of unknown origin (two of them were brothers). Theywere evaluated by flow cytometry in parallel with leukoagglutination. Reference values were established forserum reactive IgG in healthy volunteers for three dilutions (1/2, 1/5 and 1/20), both for the autologous reaction (serum and cells of the same individual) and for the heterologous reaction (serum and cells of differentindividuals). Results were expressed by an index defined by the quotient of the mean fluorescence intensityof the patientãs serum divided by that of the reference serum. Serum reactive/agglutinant factors and circulating immune complexes were evaluated in patients and parents serum. Neutrophil specific phenotypes weredetermined for HNA-1a, HNA-1b and HNA-2a. Reactive IgG/agglutinant factors were found in 4 children. Twomaternal sera were reactive against paternal and/or children neutrophils. Circulating immune complexes weredetected in 2/4 children sera and were negative in 3/3 maternal sera. Maternal/children incompatibility wasdetected in the four cases. The three mothers had the same phenotype: homozygous NA1/NA1, NB1+ (AU)

Neutropenia inmune - aloinmune neonatal IgG serica reactiva y fenotipo especifico de los neutrofilos evaluados por citometria de flujo / Autoimmune-alloimmune neonatal neutropenia. Serum reactive IgG and neutrophil-specific

La neutropenia inmune se diagnostica por la presencia de auto o aloanticuerpos reactivos con losneutrófilos. La neutropenia aloinmune neonatal es consecuencia de la sensibilización materna alos antígenos específicos de los neutrófilos paternos que afectan al neonato al atravesar la barrera placentaria. Se presentan 4 casos de niños, 2 de ellos hermanos consanguíneos con doble vínculo. Se estudiaron los sueros de los pacientes y sus padres. Por citometría de flujo se establecen los valores de referencia de la IgG sérica reactiva con los neutrófilos en voluntarios sanos, para 3 diluciones (1/2, 1/5 y 1/20) en reacción autóloga(suero y células de un mismo individuo) y heteróloga (suero y células de diferentes individuos). Los resultadosse expresan por un índice definido como el cociente entre la mediana de la intensidad de fluorescencia media del suero incógnita y la de un suero utilizado como referencia. Por leucoaglutinación se evaluó la dilucióndel suero 1/20. Se determinó el nivel de complejos inmunes circulantes. Se determinó el fenotipo, para los epitopes HNA-1a, HNA-1b y HNA-2a. En los 4 niños se encontró IgG reactiva y/o factores aglutinantes; 2/3 sueros maternos fueron reactivos con los neutrófilos del cónyuge y de los hijos. Los complejos inmunes circulantes fueron positivos en 2/4 sueros negativos en 3/3 sueros maternos. Se encontró incompatibilidad materno-infantil en los 4 casos. Las 3 madres tenían igual fenotipo: homocigotos NA1/NA1, NB1+. En síntesis, se presenta el hallazgo de 4 casos con neutropenia inmune: 3/4 auto-inmune, 1/3 se asocia a complejos inmunes circulantes y 1/4 con neutropenia neonatal aloinmune (AU)

Auto or alloantibodies reactive with neutrophils define immune neutropenia. Alloimmune neonatal neutropenia is caused by maternal sensitization to paternal neutrophil antigens, resulting in IgG antibodies that are transferred to the fetus through the placenta. We present the studies in 4 children from 3 families with neutropenia of unknown origin (two of them were brothers). Theywere evaluated by flow cytometry in parallel with leukoagglutination. Reference values were established forserum reactive IgG in healthy volunteers for three dilutions (1/2, 1/5 and 1/20), both for the autologous reaction (serum and cells of the same individual) and for the heterologous reaction (serum and cells of differentindividuals). Results were expressed by an index defined by the quotient of the mean fluorescence intensityof the patientãs serum divided by that of the reference serum. Serum reactive/agglutinant factors and circulating immune complexes were evaluated in patients and parents serum. Neutrophil specific phenotypes weredetermined for HNA-1a, HNA-1b and HNA-2a. Reactive IgG/agglutinant factors were found in 4 children. Twomaternal sera were reactive against paternal and/or children neutrophils. Circulating immune complexes weredetected in 2/4 children sera and were negative in 3/3 maternal sera. Maternal/children incompatibility wasdetected in the four cases. The three mothers had the same phenotype: homozygous NA1/NA1, NB1+ (AU)

Neutropenia inmune - aloinmune neonatal IgG serica reactiva y fenotipo especifico de los neutrofilos evaluados por citometria de flujo / Autoimmune-alloimmune neonatal neutropenia. Serum reactive IgG and neutrophil-specific

La neutropenia inmune se diagnostica por la presencia de auto o aloanticuerpos reactivos con losneutrófilos. La neutropenia aloinmune neonatal es consecuencia de la sensibilización materna alos antígenos específicos de los neutrófilos paternos que afectan al neonato al atravesar la barrera placentaria. Se presentan 4 casos de niños, 2 de ellos hermanos consanguíneos con doble vínculo. Se estudiaron los sueros de los pacientes y sus padres. Por citometría de flujo se establecen los valores de referencia de la IgG sérica reactiva con los neutrófilos en voluntarios sanos, para 3 diluciones (1/2, 1/5 y 1/20) en reacción autóloga(suero y células de un mismo individuo) y heteróloga (suero y células de diferentes individuos). Los resultadosse expresan por un índice definido como el cociente entre la mediana de la intensidad de fluorescencia media del suero incógnita y la de un suero utilizado como referencia. Por leucoaglutinación se evaluó la dilucióndel suero 1/20. Se determinó el nivel de complejos inmunes circulantes. Se determinó el fenotipo, para los epitopes HNA-1a, HNA-1b y HNA-2a. En los 4 niños se encontró IgG reactiva y/o factores aglutinantes; 2/3 sueros maternos fueron reactivos con los neutrófilos del cónyuge y de los hijos. Los complejos inmunes circulantes fueron positivos en 2/4 sueros negativos en 3/3 sueros maternos. Se encontró incompatibilidad materno-infantil en los 4 casos. Las 3 madres tenían igual fenotipo: homocigotos NA1/NA1, NB1+. En síntesis, se presenta el hallazgo de 4 casos con neutropenia inmune: 3/4 auto-inmune, 1/3 se asocia a complejos inmunes circulantes y 1/4 con neutropenia neonatal aloinmune

Auto or alloantibodies reactive with neutrophils define immune neutropenia. Alloimmune neonatal neutropenia is caused by maternal sensitization to paternal neutrophil antigens, resulting in IgG antibodies that are transferred to the fetus through the placenta. We present the studies in 4 children from 3 families with neutropenia of unknown origin (two of them were brothers). Theywere evaluated by flow cytometry in parallel with leukoagglutination. Reference values were established forserum reactive IgG in healthy volunteers for three dilutions (1/2, 1/5 and 1/20), both for the autologous reaction (serum and cells of the same individual) and for the heterologous reaction (serum and cells of differentindividuals). Results were expressed by an index defined by the quotient of the mean fluorescence intensityof the patient’s serum divided by that of the reference serum. Serum reactive/agglutinant factors and circulating immune complexes were evaluated in patients and parents serum. Neutrophil specific phenotypes weredetermined for HNA-1a, HNA-1b and HNA-2a. Reactive IgG/agglutinant factors were found in 4 children. Twomaternal sera were reactive against paternal and/or children neutrophils. Circulating immune complexes weredetected in 2/4 children sera and were negative in 3/3 maternal sera. Maternal/children incompatibility wasdetected in the four cases. The three mothers had the same phenotype: homozygous NA1/NA1, NB1+

Continuous release of hepatitis C virus (HCV) by peripheral blood mononuclear cells and B-lymphoblastoid cell-line cultures derived from HCV-infected patients.

In order to investigate hepatitis C virus (HCV) persistence and replication in peripheral blood mononuclear cells (PBMC) from a group of haemophilic individuals, HCV production and release to PBMC culture supernatants (SNs) from HCV singly infected patients and HIV/HCV co-infected patients was studied. HCV RNA+ SNs were found more frequently from HIV/HCV co-infected individuals (89.5 %) with poor reconstitution of their immune status than from singly HCV-infected patients (57 %) or from HIV/HCV co-infected individuals with a good response to highly active anti-retroviral therapy (50 %). The presence of the HCV genome in culture SNs was associated with lower CD4+ T-cell counts and with a more severe clinical picture of HIV infection. In spite of prolonged negative HCV viraemia, PBMC from HIV/HCV co-infected patients released the HCV genome after culture. HCV permissive PBMC allowed generation of HCV productive B cell lines with continuous HCV replication. These findings add further weight to the involvement of PBMCs in persistence of HCV infection and emphasize the role of B lymphocytes as HCV reservoirs.

Activation of signaling lymphocytic activation molecule triggers a signaling cascade that enhances Th1 responses in human intracellular infection.

T cell production of IFN-gamma contributes to host defense against infection by intracellular pathogens, including mycobacteria. Lepromatous leprosy, the disseminated form of infection caused by Mycobacterium leprae, is characterized by loss of cellular response against the pathogen and diminished Th1 cytokine production. Relieving bacterial burden in Ag-unresponsive patients might be achieved through alternative receptors that stimulate IFN-gamma production. We have previously shown that ligation of signaling lymphocytic activation molecule (SLAM) enhances IFN-gamma in mycobacterial infection; therefore, we investigated molecular pathways leading from SLAM activation to IFN-gamma production in human leprosy. The expression of the SLAM-associated protein (an inhibitory factor for IFN-gamma induction) on M. leprae-stimulated cells from leprosy patients was inversely correlated to IFN-gamma production. However, SLAM ligation or exposure of cells from lepromatous patients to a proinflammatory microenvironment down-regulated SLAM-associated protein expression. Moreover, SLAM activation induced a sequence of signaling proteins, including activation of the NF-kappaB complex, phosphorylation of Stat1, and induction of T-bet expression, resulting in the promotion of IFN-gamma production, a pathway that remains quiescent in response to Ag in lepromatous patients. Therefore, our findings reveal a cascade of molecular events during signaling through SLAM in leprosy that cooperate to induce IFN-gamma production and strongly suggest that SLAM might be a focal point for therapeutic modulation of T cell cytokine responses in diseases characterized by dysfunctional Th2 responses.